SUMO-1 is a 101 amino acid polypeptide that shares 18 percent sequence identity with ubiquitin. Like ubiquitin, SUMO-1 is posttransationally conjugated to a significant number of intracellular proteins. However, SUMO-1 conjugation appears to regulate the function and/or localization of targeted substrates, rather than their proteolysis. SUMO-1 conjugation has been implicated, either directly or indirectly, as a regulator of a wide range of important cell functions. Proteins identified as substrates for SUMO-1 conjugation include RanGAP1 (a regulator of nucleocytoplasmic transport), PML and Sp100 (components of intranuclear structures linked to cancer and neurodegenerative disease), and IkappaBalpha (a regulator of signaling pathways that control immune and inflammatory responses). SUMO-1 conjugation has also been implicated as a regulator of the cell cycle and DNA repair. In spite of the preliminary indications that SUMO-1 conjugation regulates a host of cell functions, the precise effects that SUMO-1 conjugation has on individual proteins, and consequently on cellular processes, remains unclear. The Specific Aims outlined in this proposal are designed to provide a more detailed understanding of SUMO- 1 conjugation, its specific effects on protein function, and its more global effects on cellular processes. These goals will be achieved through analysis of the factors and signals regulating SUMO-1 conjugation and deconjugation, characterization of novel SUMO-1 substrates, and characterization of the SUMO-1 related protein, SUMO-2. The hypothesis that SUMO-1 conjugation is regulated by E3-like protein ligases, as well as by deconjugating enzymes, will be investigated. Also, the functional relationships between SUMO-1 conjugation and PML nuclear bodies will be investigated through analysis of BLM, a novel SUMO-1/SUMO-2 substrate, and through analysis of substrates associated with the XY body of pachytene spermatocytes. The hypothesis that the XY body and PML nuclear bodies are functionally related and share common SUMO-1 conjugates will also be investigated.